This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project concerns the changes in expression of heparan sulfate (HS) structures as a function of zebra fish HS-processing enzyme knockdown. The zebra fish is an attractive system for study of the role of HS expression in mediating vasculogenesis because of the availability of mutants and the relative ease with which the phenotypes of embryos may be visualized. The aim of initial studies is to establish analytical methods for HS in this model organism. Zebra fish embryos were dechorionated at 24hpf and passed through a 30 um filter into calcium-free Ringer's solution. Cell suspensions were centrifuged, washed in PBS, and incubated in Trypsin/EDTA (0.05%) GibcoBRL) at 37[unreadable]C for 30 minutes. GAGs were isolated using a DEAE Sephacel resin Amersham Pharmacia Biotech) equilibriated with 0.2 M sodium phosphate buffer pH 6.0, washed with the same buffer and eluted with 1M NaCl containing phosphate buffer. Samples were washed and concentrated using 3 kDa cutoff Centriplus filters (Millipore, Bedford, MA). HSGAGs were digested by incubating with 3 mIU each heparinase I, heparinase II, and heparinase II (PROzyme, San Leandro, CA) at 30[unreadable]C overnight. Digestion products were analyzed using capillary electrophoresis with laser-induced fluorescence detection.